Cas9 proteins

Eupheria offers a wide range of different recombinant Streptococcus (S.) pyogenes Cas9 proteins, all of which are highly pure and effective. Fusion with a nuclear localization signal (NLS) ensures targeting of the Cas9 proteins to the nucleus. Successful genome editing by Eupheria Cas9-NLS proteins has been confirmed in a wide variety of mouse and human cell lines, and organisms, including zebrafish, Xenopus and marine plankton.

Try our Cas9-NLS proteins for your CRISPR/Cas9 experiments for greater genome editing specificity, less cell toxicity and higher insertion/deletion (indel) frequencies than with plasmid- or mRNA-encoded Cas9.

Standard scales are 10, 20, 40 and 100 µg. For pricing and other scales, please contact us.

Application Example in zebrafish

DIC = differential interference contrast; EGFP = enhanced green-fluorescent protein.
Images and results are the courtesy of Dr. Michael Brand, Center for Regenerative Therapies, Technische Universität Dresden, Germany.

 

 

Zebrafish embryos (1- to 2-cell stage) from transgenic parents, expressing EGFP-labeled myosin in the heart, were each injected with 1 ng of Cas9-NLS protein and 100 pg guide RNA targeting EGFP. Forty-eight hours post-injection, no EGFP fluorescence was detectable as a result of the Cas9-NLS-mediated knockout of the EGFP gene. In the control larvae, not injected with Cas9-NLS, the eGFP fluorescence was clearly visible in the two heart chambers.

Exemplary Cas9 in vitro activity assay

Purified recombinant Cas9-NLS protein (500 ng each) was incubated with 180 ng guide (g)RNA and 100 ng linear DNA containing the gRNA target sequence for 1 h at 37°C. The reactions were subjected to agarose gel electrophoresis. The image is courtesy of Dr. Susan Richter, Institute for Clinical Chemistry and Laboratory Medicine, Dresden,Germany.

Cas9-NLS

The Cas9-NLS represents the wild-type Cas9 nuclease from S. pyogenes, fused with a C-terminal NLS. Currently, this the best-established Cas9 protein in the field, which allows you to draw experimental advice from a large body of publications.

Cas9-Nickase-NLS

This Cas9-NLS protein contains the D10 substitution, which converts it into a nickase. The specific binding of two gRNAs in close proximity on opposite DNA strands is required for the Cas9-Nickase-NLS to cause a double-strand break. Therefore, the Cas9-Nickase-NLS provides the benefit of fewer off-targets compared with the  Cas9-NLS nuclease.

Cas9-Dead-NLS

The Cas9-Dead-NLS protein is catalytically inactive ("dead") due to the D10A and H840A substitutions. Thus Cas9-Dead-NLS binds to DNA, but does not modify it. This property allows specific genomic targeting, e. g. to block transcription.

Cas9-NLS-tagRFP

As another innovative Cas9 nuclease from Eupheria, the NLS is followed by a C-terminal tag red-fluorescent protein (tagRFP) sequence. The transfection efficiency of Cas9-NLS-tagRFP is as high as for the untagged Cas9-NLS nuclease, but the red fluorescence of successfully transfected cells allows their selection by FACS (fluorescence-activated cell sorting). The genome editing rates of such selected cell populations is 3-4 times higher than in non-selected ones.

Excitation/emission maxima:
555 nm/584 nm

Cas9-NLS-EGFP

This innovative Cas9 nuclease from Eupheria is composed of a Cas9 sequence from Streptococcus pyogenes followed by a nuclear-localization sequence (NLS) and a C-terminal enhanced green fluorescent protein (EGFP) tag. Similar to Cas9-NLS-tagRFP the transfection efficiency of Cas9-NLS-EGFP protein is as high as for the untagged Cas9-NLS nuclease. The green fluorescence allows following successfully transfected cells by e.g. fluorescence-activated cell sorting (FACS) or fluorescence microscopy. Enriching successfully transfected cells by sorting increases genome editing rates and largely reduces efforts in screening and identification of edited cells.

Excitation/emission maxima:
489 nm/509 nm

Cas9-Dead-NLS-EGFP

This Cas9 protein is catalytically inactive ("dead") due to the D10A and H840A substitutions. The NLS is followed by a C-terminal enhanced green-fluorescence protein (EGFP) sequence. The EGFP-tag offers many application possibilities, e. g. tracking of Cas9-Dead-NLS-EGFP.

Excitation/emission maxima:
488 nm/507 nm

Specifications

Production: The genes encoding the Cas9 proteins are expressed in E. coli.

NLS: The nuclear localization sequence (NLS) originates from the Simian virus 40.

Quality control: The purity of the Cas9 proteins is checked by Coomassie Blue staining after electrophoresis on SDS-polyacrylamide gels. The expected activity is confirmed in vitro and in cell culture with the mouse embryonic stem cell line E14 and the human osteosarcoma cell line U2OS.

 

Concentration: The minimum concentration is 500 ng/µL. Upon request, custom concentrations up to 5.0 µg/µL are available.

Storage and Stability: Cas9 proteins stored at -20°C are stable for at least one year. No decrease in activity was observed after 10 freeze/thaw cycles.

Storage Buffer: 20 mM HEPES pH 7.25, 150 mM KCl, 1 mM DTT.

Shipment: Shipped frozen in storage buffer at  -20°C.